In vitro RNA-Protein Binding and Pulldown Assays

In vitro RNA-protein binding and RNA pulldown assays were performed as described previously [17 (link)]. Briefly, in vitro transcribed biotinylated YIYA sense or antisense transcript were first captured by the magnetic Monoavidin beads (Bioclone Inc.) and the YIYA-bound beads were further incubated with MDA-MB-231 cell lysates. The YIYA-bound proteins/complexes were in solution digested by Immobilized Trypsin (Promega) and subjected to LC-MS/MS analysis at MD Anderson Cancer Center Proteomics Facility. For identification of binding proteins of FBXW7 and CDK6, FLAG-tagged FBXW7 and CDK6 were subjected to affinity pulldown using anti-FLAG M2 Magnetic Beads (Sigmaaldrich) using FLAG-FBXW7 and FLAG-CDK6 expressing MDA-MB-231 cells. The protein targets that associated with both FBXW7 and CDK6 were subjected to the following studies.